human mmp 13 standard mmp13  (R&D Systems)

Journal: Cancer letters

Article Title: Elevated MMP10/13 mediated barrier disruption and NF-κB activation aggravate colitis and colon tumorigenesis in both individual or full miR-148/152 family knockout mice.

doi: 10.1016/j.canlet.2021.12.033

Figure Lengend Snippet: Fig. 3. Loss of miR-148/152 family members increases MMP10 and MMP13. A. Venn diagram representing overlap of differentially expressed genes between the GEO dataset and TCGA database. B. Venn diagrams representing overlapping differentially expressed genes between miR-148/152 family target genes and upre gulated genes screened from the GEO dataset and TCGA database. C-D. Expression of MMP10 and MMP13 was detected by real-time PCR and western blotting after transfection with miR-148/152 family member inhibitors or mimics. E-F. Luciferase activity in Caco-2 cells. G. Western blotting detecting protein expression of target genes in mice. H–I. Representative IHC images of MMP10 and MMP13 immunostaining in colon sections, scale bar: 50 μm. Data in this figure are presented as mean ± SD. C, E-F analyzed via t-test.

Article Snippet: Recombinant MMP10 or MMP13 (R&D systems, USA) were diluted to 50 mg/ml in TCNB buffer (FEIYUBIO, China) with 1 mM 4-aminophenylmercuric acetate (APMA), then activated via incubating for 2 h at 37 ◦C.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Transfection, Luciferase, Activity Assay, Immunostaining

Journal: Cancer letters

Article Title: Elevated MMP10/13 mediated barrier disruption and NF-κB activation aggravate colitis and colon tumorigenesis in both individual or full miR-148/152 family knockout mice.

doi: 10.1016/j.canlet.2021.12.033

Figure Lengend Snippet: Fig. 5. Loss of miR-148/152 family members causes hyperactivation of the NF-κB signaling pathway. A. Western blotting showing MMP10 and MMP13 both shear proTNF-α to generate bioactive TNF-α fragments. B. Activation of NF-κB pathway members was examined in mice with induced colitis and CAC via western blotting. C. IHC detecting p-p65 nuclear translocation to show NF-κB signaling pathway activity, scale bar: 50 μm. D. Venn diagram for target genes in the NF-κB signaling pathway. E-F. Expression of IKKα and IKKβ detected by real-time PCR and western blotting after transfection with miR-148/152 family member inhibitors or mimics. G. Western blotting detecting protein expression of IKKα and IKKβ in mice. H. Luciferase activity in Caco-2 cells. Data in this figure are presented as mean ± SD. E, H analyzed via t-test.

Article Snippet: Recombinant MMP10 or MMP13 (R&D systems, USA) were diluted to 50 mg/ml in TCNB buffer (FEIYUBIO, China) with 1 mM 4-aminophenylmercuric acetate (APMA), then activated via incubating for 2 h at 37 ◦C.

Techniques: Western Blot, Shear, Activation Assay, Translocation Assay, Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Transfection, Luciferase

Journal: Cancer letters

Article Title: Elevated MMP10/13 mediated barrier disruption and NF-κB activation aggravate colitis and colon tumorigenesis in both individual or full miR-148/152 family knockout mice.

doi: 10.1016/j.canlet.2021.12.033

Figure Lengend Snippet: Fig. 9. Deleting the miR-148/152 family promotes colitis and colon tumorigenesis via barrier disruption and NF-κB activation. In homeostasis, miR-148/ 152 family members impede MMP10, MMP13, IKKα, and IKKβ, all of which can disrupt intestinal barrier and promote activation of the NF-κB signaling pathway. Deletion of miR-148/152 family members causes intestinal barrier dysfunction by enhancing MMP10 and MMP13, thereby increasing sensitivity to colitis. MMP10 and MMP13 activate proTNF-α, which is released during colitis to promote activation of the NF-κB signaling pathway. Accumulation of IKKα and IKKβ caused by loss of miR-148/152 family members results in hyperactivation of the NF-κB signaling pathway, which exacerbates colitis and CAC.

Article Snippet: Recombinant MMP10 or MMP13 (R&D systems, USA) were diluted to 50 mg/ml in TCNB buffer (FEIYUBIO, China) with 1 mM 4-aminophenylmercuric acetate (APMA), then activated via incubating for 2 h at 37 ◦C.

Techniques: Disruption, Activation Assay

Journal: Cell Death and Differentiation

Article Title: Crenigacestat, a selective NOTCH1 inhibitor, reduces intrahepatic cholangiocarcinoma progression by blocking VEGFA/DLL4/MMP13 axis

doi: 10.1038/s41418-020-0505-4

Figure Lengend Snippet: a Western blot analysis and semiquantitative evaluation of DLL4, VEGFA, and CD31 expression in PDX mice tissues by densitometry analysis of protein bands reveals a downregulation of DLL4, VEGFA, and CD31 protein expression in PDX mice treated with GSI. The bands were measured compared with the housekeeping GAPDH protein band, for each tissue. Average value of DLL4, VEGFA, and CD31 expression levels among all mouse treated with LY3039478 or vehicle is reported in the graph. P value showed versus vehicle treatment. Tissues PDX mice n = 10 for vehicle treatment in gray, n = 10 for LY3039478 treatment in black. b Representative images with immunofluorescence staining show DLL4 and CD31 downregulation in representative images of PDX tissues treated with LY30349478. DLL4 (green) and CD31 (red) and overlapping staining (yellow) were immunolocalized in PDX tissues. The yellow arrows highlight the detail of the co-localization of DLL4 and CD31 in PDX tissues (#4, #14, #24) not treated with LY339478. DAPI, 4′,6‐diamidino‐2‐phenylindole. c Immunofluorescence staining with MMP13 in red and nucleus in DAPI shown a significantly reduction of MMP13 in iCCA PDX tissues treated with LY3039478. Magnifications: ×20; inset ×60. d Representative images demonstrate a significant ( P < 0.001) destruction of the network created by the HUVECs following the treatment with LY3039478 (1 µM). The concomitant administration of MMP13 counteracts significantly ( P < 0.01) drug effectiveness.

Article Snippet: The recombinant protein MMP13 (R&D System, Minneapolis, MN) was used at 50 ng/ml.

Techniques: Western Blot, Expressing, Immunofluorescence, Staining

Journal: Cell Death and Differentiation

Article Title: Crenigacestat, a selective NOTCH1 inhibitor, reduces intrahepatic cholangiocarcinoma progression by blocking VEGFA/DLL4/MMP13 axis

doi: 10.1038/s41418-020-0505-4

Figure Lengend Snippet: a Analysis of 31 primary tumors from iCCA patients and matched surrounding normal liver tissues downloaded from the GEO database (GSE107943). Mean expression data were expressed in RPKM (Reads Per Kilobase Million). *** P < 0.001 calculated with Student’s t test. b NOTCH1 gene and its pro-angiogenic targets are overexpressed in human intrahepatic cholangiocarcinoma (iCCA). Levels of NOTCH1, DLL4, VEGFA, and MMP13 mRNA were significantly more elevated in iCCA ( n = 42) than corresponding nontumorous surrounding livers (SL; n = 42), as detected by quantitative reverse-transcription PCR. Number target (NT) = 2 −ΔCt , wherein ΔCt value of each sample was calculated by subtracting the average Ct value of the gene of interest from the average Ct value of the β-actin gene. Mann–Whitney test: vs SL, P < 0.0001. c Expression of the NOTCH1 gene correlates with mRNA levels of putative target genes (HES1, DLL4, VEGFA, and MMP13) in a collection of human intrahepatic cholangiocarcinoma (CCA) samples ( n = 42). Linear regression analysis was used. d Representative expression patterns of CK19, NOTCH1, HES1, DDL4, and MMP13 in human intrahepatic cholangiocarcinoma (iCCA) as detected by immunohistochemistry. Upper panels: CCA case (CCA1) showing strong, concomitant immunoreactivity for NOTCH1, HES1, DDL4, and MMP13. Lower panels: CCA specimens (CCA2) exhibiting low levels of NOTCH1, HES1, DDL4, and MMP13. As expected, both iCCA display robust immunolabeling for CK19 (a biliary marker). Magnification: ×200; scale bar = 100 μm. H&E hematoxylin and eosin staining.

Article Snippet: The recombinant protein MMP13 (R&D System, Minneapolis, MN) was used at 50 ng/ml.

Techniques: Expressing, Reverse Transcription, MANN-WHITNEY, Immunohistochemistry, Immunolabeling, Marker, Staining

Journal: Cell Death and Differentiation

Article Title: Crenigacestat, a selective NOTCH1 inhibitor, reduces intrahepatic cholangiocarcinoma progression by blocking VEGFA/DLL4/MMP13 axis

doi: 10.1038/s41418-020-0505-4

Figure Lengend Snippet: Levels of tumor microvessel density (MVD) correlate with mRNA expression of NOTCH1 ( a ), HES1 ( b ), DLL4 ( c ), and MMP13 ( e ), but not with those of VEGFA ( d ), in a collection of human intrahepatic cholangiocarcinoma (iCCA) samples ( n = 42). Linear regression analysis was used. f Representative examples of human iCCA specimens with high and low MVD.

Article Snippet: The recombinant protein MMP13 (R&D System, Minneapolis, MN) was used at 50 ng/ml.

Techniques: Expressing